New strategy for treatment of fatty liver disease

Our systems level analysis indicated that a three-step strategy including i) increasing mitochondrial fatty acid uptake, ii) increasing mitochondrial fatty acid oxidation iii) increasing the availability of GSH can be applied to decrease the amount of hepatic steatosis in NAFLD patients. Single or combined metabolic cofactors can be supplemented to boost these metabolic processes altered in NAFLD. Supplementation of i) L-carnitine (carnitine) may stimulate the transfer of fatty acids from cytosol to mitochondria, ii) NR may provide required amount of NAD+ which is essential for mitochondrial fatty acid oxidation as well as iii) serine and NAC may increase the level of GSH in the cells. The research was supported by SFO funding and the Knut and Alice Wallenberg Foundation.

Research

Background

NAFLD is a consequence of the imbalance between deposition and removal of lipids from the liver (1). The global prevalence of NAFLD continues to increase dramatically and has reached 25% at the population level (2). NAFLD includes a spectrum of pathological conditions, ranging from simple steatosis to hepatic inflammation referred as non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma (HCC) (2). Even though hepatic steatosis and fibrosis are reversible conditions, decompensated cirrhosis is frequently associated with irreversible hepatic damage, and transplantation is considered to be the only possible treatment option.

Oxidative stress and inflammatory responses have major role in the pathogenesis of NAFLD and its progression to NASH. NAFLD is also closely linked to complex metabolic conditions including obesity, type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD) (3). Considering the complexity associated with the drug development that can be used in effective treatment of the patients, supplementation of natural substances that can activate the altered metabolic pathways in NAFLD can be used in treatment of the patients. Supplementation of such metabolic cofactors may also improve the metabolic parameters in NAFLD patients and stop progression of the disease to severe form of the diseases including NASH, cirrhosis and HCC.

Underpinning research

Firstly we generated a functional liver-specific genome-scale metabolic model (GEM) (Mardinoglu et al Nature Communications, 2014 & Uhlen et al Science, 2015) and an integrated network (IN) (Lee et al Cell Metabolism, 2016) by merging GEMs with other networks. This integrative approach allows not only to reveal the key pathways, metabolites and genes involved in the progression of liver diseases, but also to make solid predictions that can be experimentally tested due to the known regulatory effect of other proteins on metabolism.

Secondly, we combined clinical studies with stable isotopes, in-depth multi-omics profiling and liver-specific networks to clarify the underlying mechanisms of NAFLD and develop strategies for prevention and treatment (Mardinoglu et al Molecular Systems Biology, 2017). To test the model-based predictions, we assessed the effect of short-term serine supplementation in NAFLD patients by providing an oral dose of 20 g of serine per day for 14 days showing that liver enzymes and plasma triglycerides, as well as the amount of fat in liver were significantly decreased after supplementation of serine. Our model also indicated that supplementation of L-carnitine as well as precursors of GSH and NAD⁺ including serine, NAC and NR would decrease liver fat accumulation by promoting the fat uptake and its oxidation in the mitochondria as well as generation of GSH required in the liver.

Thirdly, we designed a calibration study to study its toxicity and acute effect of the supplementations. We first performed a 7-day rat toxicology to study tolerability of the combined metabolic cofactors and measured clinical parameters to identify potential side effects (Zhang et al, Under review, 2019). Next, we performed a 5-day human calibration study by supplementing naturally occurring metabolic cofactors including 20 g serine, 3 g carnitine, 5 g NAC and 1 g NR and a control study to reveal the acute global effect of the supplementation of combined metabolic cofactors by eliminating the effect of the fasting. We generated plasma metabolomics and inflammatory protein markers data to reveal the changes associated with the supplementation of these metabolic cofactors.

The rational of the supplementation is that liver itself has capacity to oxidize fat stored in liver of NAFLD patients. This has been justified in our recent study, where we showed that the liver fat content is decreased more than 40% by providing high fat and protein diet during 2 weeks in NAFLD patient (Mardinoglu et al, Cell Metabolism, 2019). Based on integrative analysis, we found that the oxidation of fatty acids, de novo synthesis of GSH and catabolism of BCAAs was significantly increased whereas the consumption of glucose was significantly decreased after the supplementation of metabolic cofactors.

Finally, we observed that supplementation of these metabolic cofactors may provide a therapeutic strategy against NAFLD progression by promoting the uptake and oxidation of fat in mitochondria. Hence, we propose testing of metabolic co-factors in a randomized double-blind placebo-controlled human Phase II study to evaluate its long-term efficacy in NAFLD/NASH patients. We are currently running a multi-centre Phase 2 study in Finland and Turkey in NAFLD patients.

Details of impact

The concept that substrate deficiencies for metabolic enzymes underlie the progression of and may even be the causes of human diseases and it has rarely been mechanistically explored for different diseases. Based on our integrative systems level analysis (4), we suggested the use of carnitine, NR, serine and NAC for effective treatment of NAFLD. Of note, following the failure of recent clinical trials targeting single molecule or pathway; current NASH studies show that the trend towards targeting multiple pathways employing combination therapeutics (5). This is in line with changing our understanding of NASH pathogenesis from double-hit to multiple-hit hypothesis. Therefore, combination of supplements targeting several NASH pathways would probably be more appropriate (6).

We obtained a very good agreement between the metabolic response in liver and the predicted effect of the metabolic cofactor supplementation. This highlights the great advantage of using natural metabolites as drugs, as the effects of natural metabolites may be more predictable. Compared to other types of drugs such as small molecules and proteins, we may have much more knowledge about the interactions of metabolites in human body based on GEMs, and could predict the potential effect of such cofactors using tissue/cell specific GEMs in a systematic way. Moreover, since the metabolites are already found in our bodies, we minimize the risk of some common problems in drug discoveries such as off-targeting and solubility, and we would expect much less side effect compared to small molecules. In this context, using a natural metabolic cofactor supplementation as medication could represent a promising direction in rational drug development especially in metabolism-related diseases. Therefore, our study can be considered as a pivotal study indicating the power of this new direction in medicine.

Almost all medical drugs are foreign to the body, and their individual structures and use are largely directed to alteration of an enzyme or receptor activity, before being excreted from the body. From a toxicological point of view, using nutritional substances rarely causes safety issues in humans. The “safe doses” for most nutritional/food substances are already known in humans and, unlike most pharmaceuticals, there is often a very large margin to toxicity using nutritional compounds. As an example, the dose at which vitamin B₁₂ becomes toxic for humans is over 100-fold higher than the recommended daily intake. The reason for this low toxicity of nutritional substances is because they are generally directly or indirectly metabolized via cellular metabolic pathways. The products are easily dealt with either by shuffling substrate-overload to other metabolic pathways, or used as building blocks of other compounds (like glycogen, fat, proteins), or excreted from the cell or body.

We demonstrated that simultaneous supplementation of combined metabolic cofactors may improve the efficacy of the intervention in patients with NAFLD and decreases liver fat. Considering that NAFLD, obesity, T2D, neurodegenerative diseases and CVD are common conditions that regularly co-exist and act synergistically and it has quite similar pathogenesis with alcoholic fatty liver disease, such metabolic cofactors can also be used in the treatment of the subjects with such disorders after testing its effect in placebo-controlled human clinical studies.

References

Research

The research is conducted under the supervision of Associate Professor Adil Mardinoglu and Professor Mathias Uhlen. The research is supported by national SRA funding and the Knut and Alice Wallenberg Foundation.

1. Adil Mardinoglu, Rasmus Agren, Caroline Kampf, Anna Asplund, Mathias Uhlen and Jens Nielsen, Genome-scale metabolic modelling of hepatocytes reveals serine deficiency in patients with non-alcoholic fatty liver disease. Nature Communications, 2014. Cited: 244
2. Mathias Uhlén, Linn Fagerberg, Björn M Hallström, Cecilia Lindskog, Per Oksvold, Adil Mardinoglu, Åsa Sivertsson, Caroline Kampf, Evelina Sjöstedt, Anna Asplund, IngMarie Olsson, Karolina Edlund, Emma Lundberg, Sanjay Navani, Cristina Al-Khalili Szigyarto, Jacob Odeberg, Dijana Djureinovic, Jenny Ottosson Takanen, Sophia Hober, Tove Alm, Per-Henrik Edqvist, Holger Berling, Hanna Tegel, Jan Mulder, Johan Rockberg, Peter Nilsson, Jochen M Schwenk, Marica Hamsten, Kalle von Feilitzen, Mattias Forsberg, Lukas Persson, Fredric Johansson, Martin Zwahlen, Gunnar von Heijne, Jens Nielsen and Fredrik Ponten, A tissue-based atlas of the human proteome, Science, 2015. Cited: 3536
3. Sunjae Lee, Cheng Zhang, Murat Kilicarslan, Brian D. Piening, Elias Bjornson, Björn M. Hallström, A.K. Groen, Ele Ferrannini, Markku Laakso, Michael Snyder, Matthias Blüher, Mathias Uhlen, Jens Nielsen, Ulf Smith, Mireille J. Serlie, Jan Boren, Adil Mardinoglu, Integrated network analysis reveals an association between plasma mannose levels and insulin resistance and secretion, Cell Metabolism, 2016. Cited: 56
4. Adil Mardinoglu, Elias Bjornson, Cheng Zhang, Martina Klevstig, Sanni Söderlund, Marcus Ståhlman, Martin Adiels, Antti Hakkarainen, Nina Lundbom, Murat Kilicarslan, Björn M Hallström, Jesper Lundbom, Bruno Vergès, P Hugh R Barrett, Gerald F Watts, Mireille J. Serlie, Jens Nielsen, Mathias Uhlén, Ulf Smith, Hanns-Ulrich Marschall, Marja-Riitta Taskinen, Jan Boren, Personal model-assisted identification of NAD+ and glutathione metabolism as intervention target in NAFLD. Molecular Systems Biology, 2017. Cited: 41
5. Adil Mardinoglu, Hao Wu, Elias Bjornson, Cheng Zhang, Antti Hakkarainen, Sari M. Räsänen, Sunjae Lee, Rosellina M. Mancina, Mattias Bergentall, Kirsi H. Pietiläinen, Sanni Söderlund, Niina Matikainen, Marcus Ståhlman, Per-Olof Bergh, Martin Adiels, Brian D. Piening, Marit Granér, Nina Lundbom, Kevin J. Williams, Stefano Romeo, Jens Nielsen, Michael Snyder, Mathias Uhlén, Göran Bergström, Rosie Perkins, Hanns-Ulrich Marschall, Fredrik Bäckhed, Marja-Riitta Taskinen and Jan Borén, An Integrated Understanding of the Rapid Metabolic Benefits of a Carbohydrate-Restricted Diet on Hepatic Steatosis in Humans. Cell Metabolism, 2018. Cited: 61
6. Cheng Zhang, Elias Bjornson, Muhammad Arif, Abdellah Tebani, Alen Lovric, Rui Benfeitas, Mehmet Ozcan, Kajetan Juszczak, Woonghee Kim, Jung Tae Kim, Gholamreza Bidkhori, Marcus Ståhlman, Per-Olof Bergh, Martin Adiels, Hasan Turkez, Marja-Riitta Taskinen, Jim Bosley, Hanns-Ulrich Marschall, Jens Nielsen, Mathias Uhlén, Jan Borén, Adil Mardinoglu, The acute effect of naturally occurring metabolic cofactors supplementation. Under review in Nature Medicine, 2019.

Corroborate impact

1. Samuel, V.T. and G.I. Shulman, Nonalcoholic Fatty Liver Disease as a Nexus of Metabolic and Hepatic Diseases. Cell Metab, 2018. 27(1): p. 22-41.
2. Lonardo, A., et al., Nonalcoholic fatty liver disease: a precursor of the metabolic syndrome. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver, 2015. 47(3): p. 181-90.
3. Tilg, H., A.R. Moschen, and M. Roden, NAFLD and diabetes mellitus. Nature reviews. Gastroenterology & hepatology, 2017. 14(1): p. 32-42.
4. Mardinoglu, A., et al., Systems biology in hepatology: approaches and applications. Nat Rev Gastroenterol Hepatol, 2018. 15(6): p. 365-377.
5. Bosley, J., et al., Improving the economics of NASH/NAFLD treatment through the use of systems biology. Drug discovery today, 2017.
6. Mardinoglu, A., et al., The potential use of metabolic cofactors in treatment of NAFLD. Nutrients, 2019.